EP0184361B1 - Méthode pour activer un support polymérique - Google Patents

Méthode pour activer un support polymérique Download PDF

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EP0184361B1
EP0184361B1 EP19850308501 EP85308501A EP0184361B1 EP 0184361 B1 EP0184361 B1 EP 0184361B1 EP 19850308501 EP19850308501 EP 19850308501 EP 85308501 A EP85308501 A EP 85308501A EP 0184361 B1 EP0184361 B1 EP 0184361B1
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gel
activated
ligand
polymer
thiol
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EP0184361A3 (en
EP0184361A2 (fr
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That T. Ngo
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Bioprobe International Inc
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Bioprobe International Inc
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F8/00—Chemical modification by after-treatment
    • C08F8/30—Introducing nitrogen atoms or nitrogen-containing groups
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate

Definitions

  • This invention relates to a method of covalently binding organic ligands to polymeric carriers.
  • the invention relates to a new method of covalently binding organic ligands containing one or more primary or secondary amino or sulfhydryl groups to polymeric gels.
  • One of the first methods for immobilizing biological ligands involved treatment of a polymer containing hydroxyl groups with an activating agent such as cyanogen bromide, CNBr.
  • the activated polymer could then be used to directly bind various biological ligands to the polymer by means of covalent bonds.
  • Porath et al. describe several chemical activation methods including the CNBr method in Porath, et al., "Immobilized Enzymes. Methods in Enzymology, K. Mosbach, Ed., Vol. 44, p. 19-45, Academic Press, New York (1976).
  • Most of the early methods for activating polymers containing hydroxyl groups were subject to certain disadvantages which made their widespread use impractical.
  • CNBr activation procedures suffer from the following disadvantages: (1) the linkages formed between CNBr-activated hydroxyl containing polymers and the amino groups of ligands which are reacted with the activated polymers are labile; (2) the reaction between the activated polymer and ligand frequently results in the introduction of charged species which interfere with utilization of the reaction product in affinity absorption; (3) CNBr is a noxious, lachrimatory and poisonous chemical which requires special care in its handling.
  • the epoxide gel is then reacted with sodium thiosulfate to give a thiosulfate ester gel, which is then reduced by dithiothreitol to give a modified agarose gel containing a thiol group.
  • This so-called thiol gel is then converted to a 2-pyridyl disulfide gel by means of 2,2'-dipyridyl disulfide.
  • a solution of urease is then passed through a column of the disulfide gel to obtain an enzyme conjugate of high protein content and high catalytic activity.
  • One disadvantage of this procedure is that the epoxy substituted polymer is not stable enough to store.
  • U.S. Patent No. 4,415,665 to Mosbach et al. teaches a method of covalently binding a biologically active substance containing amino, thiol or aromatic hydroxyl groups directly to a polymeric substance containing at least one hydroxyl group by forming a reactive sulfonate derivative of the polymeric substance and then reacting the thus activated polymeric substance directly with the biologically active organic substance.
  • sulfonyl halides has proven to be advantageous in many ways, the cost of the more active organic sulfonyl halides tends to be prohibitive and tresyl chloride, being a liquid, is less convenient to handle.
  • Mukaiyama et al. discloses the use of 1-methyl-2-alkoxypyridinium salts as reagents for preparing various 2-pyridyl sulfides. Mukaiyama et al., Chem. Lett., 1159-1162 (1975).
  • a convenient method has now been found for preparing covalent chromatographic matrices utilizing a hydroxyl containing polymer which has been activated by reaction with 2-fluoro-1-methy-Ipyridinium toluene-4-sulfonate (FMP).
  • FMP 2-fluoro-1-methy-Ipyridinium toluene-4-sulfonate
  • the activated hydroxyl containing polymer can be used in forming covalent bonds with various ligands containing amino and sulfhydryl groups.
  • the covalently bound ligands are difficult to remove from the polymeric matrix. Therefore, it was found desirable to bind the ligand to the polymer in a manner such that the ligand could be readily removed when desired.
  • the procedure involves conversion of the activated polymer to a thiol gel, that is, a sulfhydryl group containing polymer.
  • the thiol gel can then be reacted with 2,2'-dipyridyl disulfidetoform a 2-pyridyl disulfide derivative of the polymer.
  • Thiol-disulfide interchange with the sulfhydryl group containing ligand causes the ligand to be linked to the polymer by means of a disulfide linkage. Removal of the ligand when desired can be readily accomplished by reduction of the disulfide linkage with a thiol such as dithiothreitol.
  • thiol gel Two different routes to the thiol gel are available.
  • sodium dimethyl dithiocarbamate is used to convert the activated polymer to the corresponding dimethyl dithiocarbamyl derivative, which, by means of reductive cleavage is converted to the desired sulfhydryl substituted polymer, hereinafter referred to as the DS-gel.
  • Another route to a thiol gel involves treatment of the FMP activated polymer with dithiothreitol to form a dithiothreityl gel, a thiol gel, hereinafter referred to as the DTT-gel, in which the free sulfhydryl group is 4 carbon atoms removed from a thioether linkage to the polymer.
  • either the DS-gel or the DTT-gel may be ideally suited for the particular chromatographic procedure which is to be carried out.
  • the DTT-gel may be particularly adapted for use in those instances where the ligand contains bulky groups which might prevent it from approaching close enough to the polymer to attack the disulfide linkage, were it notforthe space provided between the polymer surface and the disulfide linkage by the intervening 4 carbon atom chain.
  • the polymeric carrier can be a water insoluble or water soluble polymeric substance and the choice of the carrier is not critical for carrying out the process of the present invention.
  • any type of carrier can be used which has a polymeric nature and contains at least one hydroxyl group bonded to a carbon atom which is available for activation and coupling.
  • the carrier is chosen with regard to the requirements in the individual situation, primarily with regard to the type of ligand to be coupled and the intended use of the coupling product.
  • the carrier may be comprised of natural, semi-synthetic or synthetic materials containing hydroxyl groups. Examples of important carrier materials are polysaccharides and polysaccharide containing materials, for example, cellulose agarose, dextran and cross-linked products thereof.
  • Examples of synthetic carriers are polyethylene glycol, polyvinyl alcohol, polyhydroxyethyl methyl acrylate and the like. It is, of course, also possible to use carriers which normally do not contain hydroxyl groups but which, by suitable treatment, can be provided with such groups.
  • An example is silica particles, to the surface of which have been bonded groups containing at least one hydroxyl group bonded to a carbon atom.
  • hydroxyl containing polymeric carriers can be carried out in the presence of a slight excess of a tertiary amine such as triethylamine or tributylamine in dry polar organic solvents such as acetonitrile, acetone or tetrahydrofuran.
  • FMP reacts rapidly, usually in about 1-15 minutes at ambient conditions of temperature and pressure, for example, at about 22-35°C, with hydroxyl groups of various polymeric materials to form 2-alkoxy-1-methylpyridinium salts, which react readily with amino or sulfhydryl groups of various nucleophiles suitable for use as affinity ligands.
  • Other 2-halo-1-methylpyridinium salts such as 2-chloro-1-methylpyridinium salts can be used, but the 2-fluoro-1-methylpyridinium salt is preferred because of its greater reactivity.
  • Unreacted activating agent such as FMP can be easily washed from the polymeric carrier with a dilute acid such as dilute HCI, for example, with 2 mM HCI, to purify and stabilize the activated polymer without causing hydrolysis of the activated hydroxyl groups.
  • the FMP-activated polymeric carrier was found to be stable for at least 4 months when stored at 4°C in 2 mM HCI.
  • the activated polymeric carrier can also be stored in dilute mineral acids such as 2 mM phosphoric acid or in dry form, if desired. Activation densities of 4-7 micromoles per ml of gel are routinely obtained.
  • the coupling method of the present invention is generally applicable to organic ligands containing the indicated amino or sulfhydryl groups.
  • organic ligands containing the indicated amino or sulfhydryl groups may be utilized for the desired coupling to the activated polymeric hydroxyl containing polymer.
  • salts of sulfhydryl group containing compounds such as Na salts thereof are useful for this purpose.
  • the product selected for coupling should be a good nucleophile, so that the coupling reaction can be carried out smoothly. Any group capable of displacing the 1-methyl-2-pyridoxy group from the polymeric carrier is satisfactory for use as a ligand.
  • the ligand may contain any aliphatic, aromatic heterocyclic, or heteroaromatic radical or any radical which is a combination of the foregoing, so long as the resulting ligand will have functional groups available for coupling.
  • biologically active ligands for example, proteins, such as enzymes; antibodies and antigens; amino acids; thiol compounds; cofactors; nucleotides; polynucleotides; haptens and many other types of biologically active ligands, especially those having biospecific affinity to another substance which can be used, for example, for affinity chromatographic purposes.
  • reaction scheme is illustrated in Figure 1 of the drawings wherein the symbol represents a polymeric carrier having at least one -CH 27 -OH group, TsO- represents the toluene-4-sulfonate ion, TEA represents triethylamine, L-NH 2 represents an amino group containing ligand and L-SH represents a sulfhydryl group containing ligand.
  • Coupling can be carried out under varying conditions of temperature and pH and can be performed in aqueous reaction media as well as in polar organic solvents.
  • Reaction conditions are not critical for either the activation step or the coupling step and are primarily chosen with regard to the sensitivity of the reactant and to practical considerations of convenience. Mild reaction conditions are preferred. It is, for example, often suitable to work at ambient temperatures and pressures and, in the case of an aqueous reaction medium, the pH is usually close to neutral, for example, pH 8-9.
  • the degree of coupling to the hydroxyl groups of the carrier can be varied as needed by stoichionmetric adjustment to utilize essentially all available hydroxyl groups of whatever part thereof is desired. Coupling efficiencies of 70-80% are realized routinely.
  • Unreacted activated groups remaining after coupling can be removed by suspending the coupled polymer in 0.2M Tris-HCI, pH 9, at room temperature, for 2 hours.
  • Other nucleophiles such as ethanolamine or mercaptoethanol can also be used for this purpose.
  • the first step in the process shown is the reaction of hydroxyl containing polymer having at least one reactive hydroxyl group (Formula 1) with 2-fluoro-1-methylpyridinium toluene-4-sulfonate (Formula 2), which has been described above.
  • the resulting 2-alkoxy-1-methylpy- ridinium salt (Formula 3) will be referred to at times as the activated polymer or activated gel.
  • the activated polymer is readily attacked by nucleophiles, since the 1-methyl-2-pyridoxy group is a good leaving group being readily converted to 1-methyl-2-pyridone (Formula 4) upon nucleophilic substitution by a ligand.
  • Reduction of the dimethyl dithiocarbamyl derivative of the gel results in a thiol gel having a sulfhydryl group directly bonded to a carbon atom of the gel (Formula 7).
  • Reduction can be readily accomplished using any standard reducing agent such as sodium borohydride, lithium aluminum hydride or sodium dithionite.
  • the reaction takes place readily under ambient conditions of temperature and pressure and in a period of time of about 6 hours to 12 hours.
  • the resulting DS-gel has a sulfhydryl group content of 5-15 micromole per gram of dry gel.
  • thiol gel Another route to a thiol gel involves the reaction of the activated gel (Formula 3) with dithiothreitol (Formula 8) to produce the DTT-gel, (Formula 9) a thiol gel having a sulfhydryl group, which is separated by a 4 carbon atom chain from a thioether linkage to the polymer.
  • the activated gel and dithiothreitol are readily reacted by mixing in the presence of a base such as sodium bicarbonate or a tertiaryamine, such as triethylamine or tributylamine.
  • the reaction is conducted under ambient conditions of temperature and pressure and is complete in a period of about 4 to 8 hours.
  • the resulting DTT-gel like the DS-gel, can then be used as a covalent chromatographic matrix.
  • any unreacted 1-methylpy- ridoxy activating groups be removed from the DS-gel or DTT-gel in order to control the desired course of the reaction with the ligand which is the subject of the covalent chromatography. Removal of unreacted activating groups is readily accomplished by using a reactive ligand such as Tris-HCL, for example, 0.2 M Tris-HCI, pH 9, ethanolamine, mercaptoethanol or other suitable reactive ligand which will not, however, react with the sulfhydryl group of the thiol gel.
  • Tris-HCL for example, 0.2 M Tris-HCI, pH 9, ethanolamine, mercaptoethanol or other suitable reactive ligand which will not, however, react with the sulfhydryl group of the thiol gel.
  • the thiol gel is activated in order to conduct the thiol-disulfide interchange which is responsible for the binding of the desired ligand to the gel. Activation is most readily achieved by reacting the thiol gel with 2,2'-pyridyl disulfide (Formula 10). The reaction proceeds under ambient conditions of temperature and pressure and is complete in a period of about 1 to 3 hours.
  • the activated DS-gel (Formula 11) or the activated DTT-gel (Formula 12) is then reacted with the desired ligand, represented in Figure 4 as an enzyme having a free sulfhydryl group (Formula 13) in order to form a disulfide linkage between the gel and the enzyme.
  • the disulfide linkage binds the enzyme directly to a carbon atom of the gel (Formula 14), whereas in the case of the DTT-gel, the disulfide linkage binds the enzyme to a carbon atom chain linked by a sulfur atom to a carbon atom of the gel (Formula 15).
  • 2-thiopyridone (Formula 16) is displaced from the activated thiol gel.
  • Formulae 14 and 15 illustrate immobilized enzymes. It should be appreciated that many other ligands can be similarly immobilized in accordance with the above described process. Reaction of the ligand with the activated thiol gel is readily carried out under ambient conditions of temperature and pressure with suitably pufified and buffered ligand. The reaction is complete in about 2 to 6 hours.
  • the immobilized enzyme will release the enzyme (Formula 13) upon treatment with a reducing agent (Formula 17) such as cysteine, dithiothreitol or mercaptoethanol. Release of the enzyme occurs readily at ambient conditions of temperature and pressure.
  • a reducing agent such as cysteine, dithiothreitol or mercaptoethanol. Release of the enzyme occurs readily at ambient conditions of temperature and pressure.
  • Sepharose CL-4B was washed successively with 20 gel volumes of distilled water and mixtures of acetone and water having volume-to- volume ratios of 25:75, 50:50, 72:25 and 100% acetone and finally, with 10 gel volumes of dry acetone.
  • a quantity of 50 g of the washed Sepharose gel was suspended in 50 ml of dry acetonitrile mixed with 1 ml of dry triethylamine and stirred vigorously at room temperature.
  • a solution of 3 g of FMP in 40 ml of dry acetonitrile and 1.5 ml of dry triethylamine was added to the gel suspension in 5 ml portions. After 10 minutes the gel was washed with 10 gel volumes of mixtures of acetone and 2 mM HCI having a volume-to- volume ratio of 75:25, 50:50, 25:75 and undiluted 2 mM HCI.
  • the FMP-activated gel prepared according to Example 1 and stored in 2 mM phosphoric acid solution was removed from the phosphoric acid and a 20 ml quantity of the FMP-activated gel was added to 10 ml of 0.5 M NaHC0 3 containing 0.2 millimole to tobramycin.
  • the gel suspension was gently stirred at room temperature for 24 hours and then washed with 500 ml phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the washed gel was suspended in 0.1 M Tris for 15 minutes to deactivate any unreacted activated hydroxyl groups. Then the gel was washed with 500 ml of PBS, 1000 ml PBS containing 1 M NaCI, and finally with 500 ml of PBS.
  • the density of activated expressed in units of micromoles per ml of gel is measured. Since 1-methyl-2-pyridone is released from the activated gel upon coupling with nucleophiles, it is possible to quantitatively determine the density of activation by means of the absorbance at 297 nm of the solution in which the coupling reaction is conducted. At this wavelength in 0.2 M Tris-HCI, pH 9, 1-methyl-2-pyridone has a molar extinction of 5900. The density of activation can then be determined by suspending 1 ml of activated gel in 2 ml of 0.2 M Tris-HCI, pH 9, and stirring gently at room temperature for 10 hours.
  • the coupled products prepared according to the present invention are useful in various applications where it is desired to have a ligand, such as a biologically active material, immobilized by attachment to a polymeric carrier, for example, in affinity purification, covalent chromatography, and reversible and irreversible covalent immobilization of biomolecules.
  • a ligand such as a biologically active material
  • immobilized by attachment to a polymeric carrier for example, in affinity purification, covalent chromatography, and reversible and irreversible covalent immobilization of biomolecules.
  • a quantity of 1m of rabbit anti-tobramycin serum was diluted tenfold with PBS.
  • the diluted antiserum was centrifuged at 2000 rpm for 30 minutes to remove solid debris.
  • the entire supernatant was applied to a 0.5x20 cm column of Sepharose CL-4B conjugated with tobramycin prepared as described in Example 3.
  • the column was washed with PBS until the absorbance of the eluate at 280 nm was less than 0.02.
  • the antibodies were eluted with 0.1 M glycine-HCI, pH 2.5 containing 10% tetrahydrofuran in 7 ml. fractions. The results of this experiment are shown in Figure 3.
  • the ligand coupled polymeric carries prepared according to the process of this invention are useful as affinity absorbants for purifying various materials which are capable of forming affinity bonds with the ligand coupled polymer.
  • the ligand coupled polymers can be used for purification of antibodies in which case elution of the antibodies absorbed upon the affinity matrix can be accomplished without causing leakage of the ligand from the matrix.
  • the ligand coupled matrix is also characterized by stability during storage. For example, the N,e-2,4-Dinitrophenyl-L-lysine coupled Sepharose CL-4B was stored in phosphate buffered saline at 4°C without losing any ligand by leakage from the ligand-coupled matrix.
  • an important advantage of the coupling method of the present invention is that the coupled substance, that is, the ligand, is covalently bonded directly to a carbon atom of the polymeric carrier, which makes splitting off by hydrolysis unlikely. Furthermore, no additional charge is introduced during the coupling reaction as is the case in some of the prior art coupling methods. Cross-linking of the carrier material, which is a common and undesired side effect of coupling methods previously available, is avoided by means of the coupling method of the present invention.
  • Example 1 A 1 g sample of the dry activated gel of Example 1 was washed with 100 ml dry N,N-dimethylformamide (DMF) and added to 100 ml DMF containing 1.8 g sodium dimethyl dithiocarbamate. The gel suspension was stirred at room temperature for 16 hours, then washed with 100 ml dry DMF and resuspended in 50 ml dry DMF. Sodium borohydride was added to the suspension in the amount of 380 mg. The gel was continuously stirred at room temperature for 4 hours. Then another 380 mg sodium borohydride was added.
  • DMF dry N,N-dimethylformamide
  • the suspension was stirred at room temperature for 4 more hours and then the gel was washed with 200 ml DMF, 1000 ml 2 mM HCI, 500 ml 0.5 N NaCI and 500 ml 2 mM HCI.
  • the sulfhydryl content of the thiol gel was determined by means of 5,5'-dithiobis (2-nitrobenzoic acid) as described in G.L. Ellman, Arch. Biochem, Biophys, 82:70-77 (1959) and determined to be 9 micromole/g of dry gel.
  • Example 1 The dry activated gel of Example 1 in a quantity of 1 gram was added to a stirred solution of 1 M dithiothreitol (DTT) in 0.2 M NaHC0 3 . The suspension was stirred at room temperature for 5 hours. The gel was then washed with 500 ml 0.2 M NaHC03,500 ml distilled water and 1000 ml 2 mM HCI. The sulfhydryl content of the gel was found to be 6 micromole/g of dry gel.
  • DTT dithiothreitol
  • Example 6 The DS-gel of Example 6 or DTT-gel of Example 7 was washed with 60% acetone-40% 0.05 M NaHC0 3 1 mM in ethylenediaminetetraacetic acid (EDTA). The washed gel was then reacted with 0.3 M 2,2'-dipyridyl disulfide.
  • EDTA ethylenediaminetetraacetic acid
  • Partially purified jack-bean urease in the amount of 1 g was added to 40 ml 0.1 M Tris-HCI, pH 7.4 containing 1 mM EDTA and 5 mM dithiothreitol (DTT).
  • the resulting suspension was stirred at 4°C for 1 hour and then centrifuged to remove the particulate fraction.
  • a volume of 5 ml of the cloudy supernatant was passed through a Sephadex G-50C 1 x50 cm column which was equilibrated with 0.05 M Tris-HCI, pH 7.4 containing 0.5 mM EDTA. This procedure insured that any dithiothreitol present would be removed in order to prevent interference during the subsequent covalent chromatographic procedure.
  • a 5 ml volume of the DTT-free eluate was applied to a 0.5x20 cm column of the activated DS-gel of Example 8.
  • the column was then washed with 0.05 M Tris-HCI, pH 7.4, containing 0.5 mM EDTA until the absorbance at 280 nm of the eluate was less than 0.1 and with 0.05 M Tris-HCI, pH 7.4 containing 0.05 mM EDTA and 0.05 M NaCi until the absorbance was less than 0.02.
  • the enzyme urease was eluted from the column by 0.05 mM Tris-HCI, pH 7.4, containing 0.05 mM EDTA and 20 mM dithiothreitol (DTT).
  • Urease activity was assayed by measuring the rate of disappearance of NADH absorbance at 340 nm through a glutamic dehydrogenase coupled reaction.
  • the substrate solution used in the reaction contained 0.05 M Tris-HCI, pH 7.4; 0.5 mM EDTA; 1 mM ADP; 1 mM alpha-ketoglutarate; 50 mM urea and 50U glutamic dehydrogenase.
  • the assay was initiated by adding 5-10 microliters of enzyme solution to 2 ml of substrate solution. The results of the immobilization of the urease upon the column and the elution from the column are shown in Figure 5.
  • UV absorbing material constituted mostly 2-thiopyridone (approximately 90%) was established by dialysis of these fractions against 2000 ml 0.05 mM EDTA in 0.05 M Tris-HCI, pH 7.4, which resulted in an average 34-fold reduction in their UV absorbance.
  • Urease activity also increased at the same time as the increase in UV absorbance.
  • a single passage of the enzyme solution through the activated thiol gel resulted in an 11-fold purification with 83% recovery of the total enzyme.
  • the purified enzyme was found to have a specific activity of 770 units/mg.
  • the following example illustrates the reversible immobilization of a biologically active material by means of covalent linkage through a disulfide bridge to the activated thiol gel of the present invention. It should be pointed out that immobilization of a biologically active material for the purpose of providing such material in a form which is convenient to use requires that the disulfide bridge be formed utilizing a sulfhydryl group which is not essential to the activity of the biologically active material.
  • E. Coli beta-galactosidase in a quantity of 5 mg was dissolved in 10 ml of 0.1 M sodium phosphate buffer, 0.15 M in NaCI, pH 7.4.
  • Wet activated DTT-gel prepared according to the procedure of Example 8 quantity of 2.5 g was added to the enzyme solution.
  • the resulting suspension was stirred at room temperature. Attimed intervals, 0.1 ml of the gel suspension was withdrawn and centrifuged at 2500 rpm for 1 minute.
  • the supernatant was diluted 50x with phosphate buffer and 0.25 ml of the diluted supernatant was assayed for beta-galactosidase.in 2 ml o-nitrophenyl-beta-D-galactopyranoside solution. After 4 hours incubation at room temperature total immobilization of the beta-galactosidase with full activity was realized. At that time 20 mM DTT in 0.05 M phosphate, pH 7.7, was mixed with the immobilized enzyme and samples were withdrawn and assayed for beta-galactosidase. As shown in Figure 6, the activity of beta-galactosidase increased until at 8 hours reaction time a total recovery of enzyme was realized.

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Claims (2)

1. Procédé d'activation d'un groupe hydroxyle d'une substance polymère comportant au moins un groupe hydroxyle qui consiste:
à faire réagir une substance polymère comportant au moins un groupe hydroxyle sur du toluène-4-sulfonate de 2-fluoro-1-méthylpyridi- nium, et
à recueillir un produit polymère, au moins certains des groupes hydroxyle du polymère ayant été transformés en groupe 1-méthyl-2-pyri- doxy.
2. Procédé de liaison par covalence de:
(A) un ligand organique qui comporte au moins un substituant choisi parmi les groupes amine primaire, amine secondaire et les groupes sulfhydryle;
directement à une:
(B) substance polymère qui comporte au moins un groupe hydroxyle; caractérisé en ce qu'il consiste
(1) à former d'abord un dérivé réactif par réaction de:
(i) toluène-4-sulfonate de 2-fluoro-1-méthylpyri- dinium sur
(ii) une substance polymère comportant au moins un groupe hydroxyle, au moins un groupe hydroxyle réactif étant relié à des atomes de carbone de la substance polymère, puis
(2) à faire réagir ce dérivé réactif directement sur le ligand organique tel qu'indiqué en (A).
EP19850308501 1984-12-07 1985-11-22 Méthode pour activer un support polymérique Expired EP0184361B1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AT85308501T ATE59658T1 (de) 1984-12-07 1985-11-22 Verfahren zur aktivierung eines polymeren traegers.

Applications Claiming Priority (4)

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US67916784A 1984-12-07 1984-12-07
US679167 1984-12-07
US679525 1984-12-07
US06/679,525 US4582875A (en) 1984-12-07 1984-12-07 Method of activating hydroxyl groups of a polymeric carrier using 2-fluoro-1-methylpyridinium toluene-4-sulfonate

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EP0184361A2 EP0184361A2 (fr) 1986-06-11
EP0184361A3 EP0184361A3 (en) 1987-11-04
EP0184361B1 true EP0184361B1 (fr) 1991-01-02

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US4753983A (en) * 1986-05-07 1988-06-28 Bioprobe International, Inc. Polymeric matrix for affinity chromatography and immobilization of ligands
GB2267502B (en) * 1992-05-28 1997-01-22 Aligena Ag Polymeric reaction products for use in immobilized buffered gels and membranes
MX237931B (es) 1998-11-30 2006-06-21 Gottschall Instruction Dr Proceso para la preparacion de una red polimerica.
DE19855173C2 (de) 1998-11-30 2001-03-15 Gottschall Instruction Ges Fue Verfahren zur Herstellung derivatisierter Polymere und Derivate von funktionelle Gruppen aufweisende Polymere sowie Verfahren zur Substratbindung
JP3774888B2 (ja) * 2001-11-28 2006-05-17 日本化薬株式会社 ステロイド類化合物のｌｃ−ｍｓによる高感度検出法
US8980238B2 (en) * 2009-09-30 2015-03-17 Thiomatrix Forschungs—Und Beratungs GmbH Mucoadhesive polymers having vitamin B partial structures

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US4195128A (en) * 1976-05-03 1980-03-25 Bayer Aktiengesellschaft Polymeric carrier bound ligands
US4415665A (en) * 1980-12-12 1983-11-15 Pharmacia Fine Chemicals Ab Method of covalently binding biologically active organic substances to polymeric substances
NZ213819A (en) * 1984-10-31 1988-05-30 Kanegafuchi Chemical Ind Lipoprotein adsorbent using hydroxy-containing polymer gel

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JPH06340707A (ja) 1994-12-13
JPH074240B2 (ja) 1995-01-25
EP0184361A3 (en) 1987-11-04
EP0184361A2 (fr) 1986-06-11
AU581947B2 (en) 1989-03-09
JPH0747602B2 (ja) 1995-05-24
CA1239357A (fr) 1988-07-19
JPH0747603B2 (ja) 1995-05-24
DE3581069D1 (de) 1991-02-07
JPS61173778A (ja) 1986-08-05
JPH06340708A (ja) 1994-12-13

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